Cloned genes encoding reverse transcriptase lacking RNase H activity

ABSTRACT

The invention relates to a gene which encodes reverse transcriptase having DNA, polymerase activity and substantially no RNase H activity. The invention also relates to vectors containing the gene and hosts transformed with the vectors of the invention. The invention, also relates to a method of producing reverse transcriptase having DNA polymerase activity and substantially no RNase H activity by expressing the reverse transcriptase genes of the present invention in a host. The invention also relates to a method of producing cDNA from mRNA, using the reverse transcriptase of the invention. The invention also relates to a kit for the preparation of cDNA from mRNA comprising the reverse transcriptase of the invention.

FIELD OF THE INVENTION

The invention is in the field of recombinant genetics.

BACKGROUND OF THE INVENTION

Both viral and cloned reverse transcriptase (RT) contain at least twoenzymatic activities, DNA polymerase and ribonuclease H(RNase H) thatreside on a single polypeptide. Grandgenett, D. P. et al., Proc. Natl.Acad. Sci. (USA) 70:230-234 (1973); Moelling, K., Virology 62:46-59(1974); Kotewicz, H. L., et al., Gene 35:249-258 (1985); and Roth, H.J., et al., J. Biol. Chem. 260:9326-9335 (1985). Little is known aboutthe structure-functional relationship of these two activities, but suchknowledge would be important both in understanding retroviralreplication and in exploiting the enzyme as a recombinant DNA tool.

In the retrovirus life cycle, the RT DNA polymerase activity isresponsible for transcribing viral RNA into double-stranded DNA. Varmus,H. (1982), in Weiss, R., et al. (eds.), RNA Tumor Viruses, Cold SpringHarbor Laboratory, pp. 410-423. The function of RNase H in replicationis less clear, but it is thought to degrade genomic RNA during DNAsynthesis to generate oligomeric RNA primers for plus-strand DNAsynthesis, and to remove the RNA primers of both minus- and plus-strandDNA. Omer, C. A., et al., Cell 30:797-805 (1982), Resnick, R., et al.,J. Virol. 51:813-821 (1984); Varmus, H. (1985), in Weiss, R., et al.(eds.), RNA Tumor Viruses, Cold Spring Harbor Laboratory, pp. 79-80.

The temporal relationship in vivo between DNA polymerization and RNAhydrolysis is not well defined. Furthermore, precisely how the twoenzymatic activities are coordinated is not clear. Conditional mutationsrestricted to either DNA polymerase or RNase H would be invaluable indeciphering, the events of retroviral replication. Unfortunately,conditional viral mutations in the RT gene invariably affect bothactivities. Lai, M. H. T, et al., J. Virol. 27:823-825 (1978); Moelling,K., et al., J. Virol. 32:370-378 (1979).

RT is used extensively in recombinant DNA technology to synthesize cDNAfrom mRNA. One major problem with cDNA synthesis is that the RNase Hactivity of RT degrades the mRNA template during first-strand synthesis.The mRNA poly(A)-oligo(dT) hybrid used as a primer for first-strand cDNAsynthesis is degraded by RT RHase H. Thus, at the outset of cDNAsynthesis, a competition is established between RNase H-mediateddeadenylation of mRNA and initiation of DNA synthesis, which reduces theyield of cDNA product. Berger, S. L., et al., Biochem. 22:2365-2373(1983). Furthermore, in some cases, the RNase H causes prematuretermination of DNA chain growth. Unfortunately, these events eliminatethe potential for repeated copying of the RNA template.

Efforts to selectively inactivate RT RNase H with site-specificinhibitors have been unsuccessful (for review, see Gerard, G. F. (1983),in Jacob, S. T., (ed.), Enzymes of Nucleic Acid Synthesis andModification, Vol. 1, DNA Enzymes, CRC Press, Inc., Coca Raton, Fla.,pp. 1-38). Attempts to physically separate the active centers of RTpolymerase and RNase H activity by proteolysis have yielded aproteolytic fragment possessing only RNase H activity (Lai, M. H. T., etal., J. Virol. 25:652-663 (1978); Gerard, G. F., J. Virol. 26:16-28(1978); and Gerard, G. F., J. Virol. 37:748-754 (1981)), but nocorresponding fragment containing only polymerase activity has beenisolated.

Computer analysis of the amino acid sequences from the putative geneproducts of retroviral pol genes has revealed a 150-residue segment atthe carboxyl terminus that is homologous with the ribonuclease H of E.coli and a section close to the amino terminus which can be aligned withnonretroviral polymerases. Johnson, M. S., et al., Proc. Natl. Acad.Sci. (USA) 83:7648-7652 (1986). Based on these related amino acidsequences, Johnson et al. suggest that ribonuclease H activity should besituated at the carboxyl terminus, and the DNA polymerase activity atthe amino terminus.

There have been a number of reports concerning the cloning of geneswhich encode RT and their expression in hosts. Weiss et al., U.S. Pat.No. 4,663,290 (1987); Gerard, G. F., DNA 5:271-279 (1986); Kotewicz, M.L., at al., Gene 35:249-258 (1985); Tanese, N., et al., Proc. Natl.Acad. Sci. (USA) 82:4944-4948 (1985); and Roth, M. J., et al., J. Biol,Chem. 260:9326-9335 (1985).

There has been no direct scientific evidence that amino acid residuesinvolved catalytically or structurally in the RNase H activity ofreverse transcriptase could be altered to eliminate RNase H activitywithout affecting the RNA-dependant DNA polymerase activity of reversetranscriptase. Moreover, there has been no report of the cloning of RTto give a gene product without RNase activity.

SUMMARY OF THE INVENTION

The invention relates to a gene which encodes reverse transcriptasehaving DNA polymerase activity and substantially no RNase H activity.

The invention also relates to a reverse transcriptase gene comprisingthe following DNA sequence:                                                                           1078ATG ACC CTA AAT ATA GAA GAT GAG CAT CGG CTA CAT GAG ACC TCA AAA GAG CCAGAT GTT MET Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser LysGlu Pro Asp Val                                                                           1138TCT CTA GGG TCC ACA TGG CTG TCT GAT TTT CCT CAG GCC TGG GCG GAA ACC GGGGGC ATG Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala GluThr Gly Gly MET                                                                           1198GGA CTG GCA GTT CGC CAA GCT CCT CTG ATC ATA CCT CTG AAA GCA ACC TCT ACCCCC GTG Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala ThrSer Thr Pro Val                                                                           1258TCC ATA AAA CAA TAC CCC ATG TCA CAA GAA GCC AGA CTG GGG ATC AAG CCC CACATA CAG Ser Ile Lys Gln Typ Pro MET Ser Gln Glu Ala Arg Leu Gly Ile LysPro His Ile Gln                                                                           1318AGA CTG TTG GAC CAG GGA ATA CTG GTA CCC TGC CAG TCC CCC TGG AAC ACG CCCCTG CTA Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cyn Gln Ser Pro Trp AsnThr Pro Leu Leu                                                                           1378CCC GTT AAG AAA CCA GGG ACT AAT GAT TAT AGG CCT GTC CAG GAT CTG AGA GAAGTC AAC Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp LeuArg Glu Val Asn                                                                           1438AAG CGG GTG GAA GAC ATC CAC CCC ACC GTG CCC AAC CCT TAC AAC CTC TTG AGCGGG CTC Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn LeuLeu Ser Gly Leu                                                                           1498CCA CCG TCC CAC CAG TGG TAC ACT GTG CTT GAT TTA AAG GAT GCC TTT TTC TGCCTG AGA Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala PhePhe Cys Leu Arg                                                                           1558CTC CAC CCC ACC AGT CAG CCT CTC TTC GCC TTT GAG TGG AGA GAT CCA GAG ATGGGA ATC Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp ProGlu MET Gly Ile                                                                           1618TCA GGA CAA TTG ACC TGG ACC AGA CTC CCA CAG GGT TTC AAA AAC AGT CCC ACCCTG TTT Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn SerPro Thr Leu Phe                                                                           1678GAT GAG GCA CTG CAC AGA GAC CTA GCA GAC TTC CGG ATC CAG CAC CCA GAC TTGATC CTG Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His ProAsp Leu Ile Leu                                                                           1738CTA CAG TAC GTG GAT GAC TTA CTG CTG GCC GCC ACT TCT GAG CTA GAC TGC CAACAA GGT Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu AspCys Gln Gln Gly                                                                           1798ACT CGG GCC CTG TTA CAA ACC CTA GGG AAC CTC GGG TAT CGG GCC TCG GCC AAGAAA GCC Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala SerAla Lys Lys Ala                                                                           1858CAA ATT TGC CAG AAA CAG GTC AAG TAT CTG GGG TAT CTT CTA AAA GAG GGT CAGAGA TGG Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys GluGly Gln Arg Trp                                                                           1918CTG ACT GAG GCC AGA AAA GAG ACT GTG ATG GGG CAG CCT ACT CCG AAG ACC CCTCGA CAA Leu Thr Glu Ala Arg Lys Glu Thr Val MET Gly Gln Pro Thr Pro LysThr Pro Arg Gln                                                                           1978CTA AGG GAG TTC CTA GGG ACG GCA GGC TTC TGT CGC CTC TGG ATC CCT GGG TTTGCA GAA Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile ProGly Phe Ala Glu                                                                           2038ATG GCA GCC CCC TTG TAC CCT CTC ACC AAA ACG GGG ACT CTG TTT AAT TGG GGCCCA GAC MET Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe AsnTrp Gly Pro Asp                                                                           2098CAA CAA AAG GCC TAT CAA GAA ATC AAG CAA GCT CTT CTA ACT GCC CCA GCC CTGGGG TTG Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala ProAla Leu Gly Leu                                                                           2158CCA GAT TTG ACT AAG CCC TTT GAA CTC TTT GTC GAC GAG AAG CAG GGC TAC GCCAAA GGT Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln GlyTyr Ala Lys Gly                                                                           2218GTC CTA ACG CAA AAA CTG GGA CCT TGG CGT CGG CCG GTG GCC TAC CTG TCC AAAAAG CTA Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr LeuSer Lys Lys Leu                                                                           2278GAC CCA GTA GCA GCT GGG TGG CCC CCT TGC CTA CGG ATG GTA GCA GCC ATT GCCGTA CTG Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg MET Val Ala AlaIle Ala Val Leu                                                                           2338ACA AAG GAT GCA GGC AAG CTA ACC ATG GGA CAG CCA CTA GTC ATT CTG GCC CCCCAT GCA Thr Lys Asp Ala Gly Lys Leu Thr MET Gly Gln Pro Leu Val Ile LeuAla Pro His Ala                                                                           2398GTA GAG GCA CTA GTC AAA CAA CCC CCC GAC CGC TGG CTT TCC AAC GCC CGG ATGACT CAC Val Glu Ala Leu Val Lys Gln Pro Pro Asp Arg Trp Leu Ser Asn AlaArg MET Thr His                                                                           2458TAT CAG GCC TTG CTT TTG GAC ACG GAC CGG GTC CAG TTC GGA CCG GTG GTA GCCCTG AAC Tyr Gln Ala Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro ValVal Ala Leu Asn                                                                   2512CCG GCT ACG CTG CTC CCA CTG CCT GAG GAA GGG CTG CAA CAC AAC TGC CTT GATPro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu Aspor the degenerate variants thereof.

The invention also relates to a reverse transcriptase gene comprisingthe following DNA sequence:                                                                           1078ATG ACC CTA AAT ATA GAA GAT GAG CAT CGG CTA CAT GAG ACC TCA AAA GAG CCAGAT GTT MET Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser LysGlu Pro Asp Val                                                                           1138TCT CTA GGG TCC ACA TGG CTG TCT GAT TTT CCT CAG GCC TGG GCG GAA ACC GGGGGC ATG Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala GluThr Gly Gly MET                                                                           1198GGA CTG GCA GTT CGC CAA GCT CCT CTG ATC ATA CCT CTG AAA GCA ACC TCT ACCCCC GTG Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala ThrSer Thr Pro Val                                                                           1258TCC ATA AAA CAA TAC CCC ATG TCA CAA GAA GCC AGA CTG GGG ATC AAG CCC CACATA CAG Ser Ile Lys Gln Tyr Pro MET ser Gln Glu Ala Arg Leu Gly Ile LysPro His Ile Gln                                                                           1318AGA CTG TTG GAC CAG GGA ATA CTG GTA CCC TGC CAG TCC CCC TGG AAC ACG CCCCTG CTA Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp AsnThr Pro Leu Leu                                                                           1378CCC GTT AAG AAA CCA GGG ACT AAT GAT TAT AGG CCT GTC CAG GAT CTG AGA GAAGTC AAC Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp LeuArg Glu Val Asn                                                                           1438AAG CGG GTG GAA GAC ATC CAC CCC ACC GTG CCC AAC CCT TAC AAC CTC TTG AGCGGG CTC Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn LeuLeu Ser Gly Leu                                                                           1498CCA CCG TCC CAC CAG TGG TAC ACT GTG CTT GAT TTA AAG GAT GCC TTT TTC TGCCTG AGA Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala PhePhe Cys Leu Arg                                                                           1558CTC CAC CCC ACC AGT CAG CCT CTC TTC GCC TTT GAG TGG AGA GAT CCA GAG ATGGGA ATC Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp ProGlu MET Gly Ile                                                                           1618TCA GGA CAA TTG ACC TGG ACC AGA CTC CCA CAG GGT TTC AAA AAC AGT CCC ACCCTG TTT Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn SerPro Thr Leu Phe                                                                           1678GAT GAG GCA CTG CAC AGA GAC CTA GCA GAC TTC CGG ATC CAG CAC CCA GAC TTGATC CTG Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His ProAsp Leu Ile Leu                                                                           1738CTA CAG TAC GTG GAT GAC TTA CTG CTG GCC GCC ACT TCT GAG CTA GAC TGC CAACAA GGT Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu AspCys Gln Gln Gly                                                                           1798ACT CGG GCC CTG TTA CAA ACC CTA GGG AAC CTC GGG TAT CGG GCC TCG GCC AAGAAA GCC Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala SerAla Lys Lys Ala                                                                           1858CAA ATT TGC CAG AAA CAG GTC AAG TAT CTG GGG TAT CTT CTA AAA GAG GGT CAGAGA TGG Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys GluGly Gln Arg Trp                                                                           1918CTG ACT GAG GCC AGA AAA GAG ACT GTG ATG GGG CAG CCT ACT CCG AAG ACC CCTCGA CAA Leu Thr Glu Ala Arg Lys Glu Thr Val MET Gly Gln Pro Thr Pro LysThr Pro Arg Gln                                                                           1978CTA AGG GAG TTC CTA GGG ACG GCA GGC TTC TGT CGC CTC TGG ATC CCT GGG TTTGCA GAA Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile ProGly Phe Ala Glu                                                                           2038ATG GCA GCC CCC TTG TAC CCT CTC ACC AAA ACG GGG ACT CTG TTT AAT TGG GGCCCA GAC MET Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe AsnTrp Gly Pro Asp                                                                           2098CAA CAA AAG GCC TAT CAA GAA ATC AAG CAA GCT CTT CTA ACT GCC CCA GCC CTGGGG TTG Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala ProAla Leu Gly Leu                                                                           2158CCA GAT TTG ACT AAG CCC TTT GAA CTC TTT GTC GAC GAG AAG CAG GGC TAC GCCAAA GGT Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln GlyTyr Ala Lys Gly                                                                           2218GTC CTA ACG CAA AAA CTG GGA CCT TGG CGT CGG CGG GTG GCC TAC CTG TCC AAAAAG CTA Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr LeuSer Lys Lys Leu                                                                           2278GAC CCA GTA GCA GCT GGG TGG CCC CCT TGC CTA CGG ATG GTA GCA GCC ATT GCCGTA CTG Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg MET Val Ala AlaIle Ala Val Leu                                                                           2338ACA AAG GAT GCA GGC AAG CTA ACC ATG GGA CAG CCA CTA GTC ATT CTG GCC CCCCAT GCA Thr Lys Asp Ala Gly Lys Leu Thr MET Gly Gln Pro Leu Val Ile LeuAla Pro His Ala                                                                           2398GTA GAG GCA CTA GTC AAA CAA CCC CCC GAC CGC TGG CTT TCC AAC GCC CGG ATGACT CAC Val Glu Ala Leu Val Lys Gln Pro Pro Asp Arg Trp Leu Ser Asn AlaArg MET Thr His                                                                           2458TAT CAG GCC TTG CTT TTG GAC ACG GAC CGG GTC CAG TTC GGA CCG GTG GTA GCCCTG AAC Tyr Gln Ala Leu Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro ValVal Ala Leu Asn                                                                           2518CCG GCT ACG CTG CTC CCA CTG CCT GAG GAA GGG CTG CAA CAC AAC TGC CTT GATAAT TCC Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn CysLeu Asp Asn Ser            2530 CGC TTA ATT AAT Arg Leu Ile Asnor the degenerate variants thereof.

The invention also relates to the vectors containing the gene of theinvention, hosts transformed with the vectors of the invention, and thereverse transcriptase expressed by the transformed hosts of theinvention.

The invention also relates to a fusion protein comprising a polypeptidehaving RNA-dependent DNA polymerase activity and substantially no RNaseH activity and a second peptide selected from polypeptide proteins whichstabilize the fusion protein and hydrophobic leader sequences.

The invention also relates to a method of producing reversetranscriptase having DNA polymerase activity and substantially no RNaseH activity, comprising culturing transformed hosts of the inventionunder conditions which produce reverse transcriptase, and isolating thereverse transcriptase so produced.

The invention also relates to a method of preparing cDNA from mRNAcomprising contacting mRNA with a polypeptide having RNA-dependent DNApolymerase activity and substantially no RNase H activity, and isolatingthe cDNA so produced.

The invention also relates to a kit for the preparation of cDNA frommRNA comprising a carrier being compartmentalized to receive in closeconfinement therein one or more containers, wherein

-   -   (a) a first container contains reverse transcriptase having DNA        polymerase activity and substantially no RNase H activity;    -   (b) a second container contains a buffer and the nucleoside        triphosphates;    -   (c) a third container contains oligo(dT)primer; and    -   (d) a fourth container contains control RNA.

The invention is related to the discovery that portions of the RT genecan be deleted to give a deletion mutant having DNA polymerase activitybut no detectable. RNase H activity. This purified mutant RT lackingRNase H activity can be used to effectively synthesize cDNA from mRNA.

DESCRIPTION OF THE FIGURES

FIG 1. This figure depicts the restriction map of plasmid pRT601. TheM-MLV RT gene extends from position 1,019 to 3,070.

FIG. 2. This figure depicts schematic representation of pRT601 andrelated plasmids, and the enzymatic activities and predicted structureof the M-MLV RT protein coded by each plasmid.

FIG. 3. This figure depicts an SDS-polyacrylamide gel of M-MLV RT.pRTdEcoRV-C RT (A) and pRT601 RT (B) (3 μg of each) were run on an SDS10% polyacrylamide gel (Laemmli, U. K., Nature 227:680-685 (1970)). Thegel was stained with Coomassie blue. Lane M contained Kr standards.

FIG. 4. This figure depicts an autoradiogram of ³²P-labeled cDNAsynthesized from 6.2 kb RNA (Materials and Methods) by pRTdEcoRV-C RT(A) or pRT601 RT (B). A 1 kb ladder was used as a standard (C).Electrophoresis was performed on an alkaline 1.4% agarose gel (McDonnel,M. W., et al., J. Mol. Biol. 110:119-146 (1977)).

FIG. 5. This figure depicts an autoradiogram of ³²P-labeled 2.3 kbpoly(A)-tailed RNA after oligo(dT)-primed cDNA synthesis catalyzed bypRTdEcoRV-C-RT or pRT601 RT. Aliquots were removed from reactionmixtures containing no enzyme (−E) or 200 units of RT at the timesindicated (in min). The minus enzyme control was incubated for 60 min.Samples were electrophoresed as described in Materials and Methods. A 1kb ladder was used as marker (M).

FIG. 6. This figure depicts the DNA sequence which encodes reversetranscriptase having DNA polymerase activity and substantially no RNaseH activity. Also shown is the corresponding amino acid sequence.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the production of reverse transcriptase havingDNA polymerase activity and substantially no RNase H activity, usingrecombinant DNA techniques.

Recombinant plasmids constructed as described herein provide reversetranscriptase for use in recombinant. DNA technology to synthesize cDNAfrom mRNA without the problem associated with RNase H activity whichdegrades mRNA template during first-strand synthesis.

By the terms “substantially no RNase H activity” is intended reversetranscriptase purified to near homogeneity and having an RNase Hactivity of less than 0.001 pmoles [³H](A)_(n) solubilized per μgprotein with a [³H](A)_(n)+(dT)_(n) substrate in which the [³H](A)_(n)has a specific radioactivity of 2,200 cpm/pmole. RNase H activities ofthis specific activity or less allows the preparation of cDNA withoutsignificant degradation of the mRNA template during first-strandsynthesis.

By the terms “degenerate variants” is intended cloned genes havingvariations of DNA sequence, but which encode the same amino acidsequence.

The reverse transcriptase gene (or the genetic information containedtherein) can be obtained from a number of different sources. Forinstance, the gene may be obtained from eukaryotic cells which areinfected with retrovirus, or from a number of plasmids which containeither a portion of or the entire retrovirus genome. In, addition,messenger RNA-like RNA which contains the RT gene can be obtained fromretroviruses. Examples of sources for RT include, but are not limitedto, Moloney murine leukemia virus (M-MLV); human T-cell leukemia virustype I (HTLV-I); bovine leukemia virus (BLV); Rous Sarcoma virus (RSV);human immunodeficiency virus (HIV); yeast, including Saccharomyces,Neurospora, Drosophila; primates; and rodents. See, for example, Weisset al., U.S. Pat. No. 4,663,290 (1987); Gerard, G. R., DNA 5:271-279(1986); Kotewicz, M. L., et al., Gene 35:249-258 (198051 Tanese, N., etal., Proc. Natl. Acad. Sci. (USA) 82:4944-4948 (1985) Rothe M. J., etal. J. Biol. Chem. 260:9326-9335 (1985). Michel, F., et al., Nature316:641-643 (1985); Akins, R. A., et al., Cell 47: 505-516 (1986), EMBOJ. 4:1267-1275 (1985); and Fawcett, D. F., Cell 47:1007-1015 (1986).

RT proviral DNA can be isolated using standard isolation techniques. TheDNA is cleaved into linear fragments, any one of which may contain thegenes which encode RT. Such fragmentation can be achieved using enzymeswhich digest or cleave DNA, such as restriction enzymes which cleave DNAas specific base sequences. After the linear DNA fragments aregenerated, they are separated according to size by standard techniques.Such recombinant DNA techniques may be performed as described byManiatis, T., et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).

Identification of the DNA fragment which contains the gene may beaccomplished in a number of ways. For example, it is possible tosequence the DNA fragments (Maxam and Gilbert, Methods in Enzymology64:499. (1980); Messing, J., Meth. in Enz. 101C:20 (1983)) to identifywhich fragment contains the reverse transcriptase gene. Alternatively,hybridization techniques (Southern, J. Mol. Biol. 98:503 (1975)) using alabeled (e.g., radioactively labeled) DNA probe may be employed.

The fractions containing the desired DNA are pooled, ligated into asuitable vector, and used to transform a host. Screening for transformedhosts containing the RT gene may be accomplished by, for example, themethod disclosed by Gerard et al., Biochem. 13:1632-1641 (1974) or byGerard et al., J. Virol. 15:785-797 (1975). Alternatively, clonescontaining reverse transcriptase may be identified by hybridization withcomplementary labeled DNA.

An alternative to isolating the reverse transcriptase gene from aretroviral proviral DNA is to make cDNA to the mRNA-like RNA which codesfor reverse transcriptase. To this end, mRNA-like RNA coding for reversetranscriptase is isolated from retrovirus. By standard techniques, theisolated mRNA is then converted into cDNA using reverse transcriptase.The cDNA can then be inserted into a plasmid vector in a conventionalmanner.

The choice of a suitable vector depends on a number of considerationsknown to one of ordinary skill in the art, such as the size of thefragment, nature of the host, number and position of restriction sitesdesired, and the selection marker and markers desired. Such vectors mayinclude replicon and control sequences from species compatible with ahost cell (see Maniatis et al., supra). Expression of the RT genes mayalso be placed under control of other regulatory sequences homologous orheterologous to the host organism in its untransformed state. Forexample, lactose-dependent E. coli chromosomal DNA comprises a lactoseor lac operon which mediates lactose utilization by elaborating theenzyme β-galactosidase. The lac control elements may be obtained frombacteriophage lambda plac 5, which is infectious for E. coli. The lacpromoter-operator system can be induced by IPTG.

Other promoter-operator systems or portions thereof can be employed aswell. For example, galactose, alkaline phosphatase, tryptophan, xylose,tac, lambda pL, lambda pR and the like can be used. Once the vector orDNA sequence containing the constructs has been prepared, the vectorsmay be introduced into an appropriate host. Various techniques may beemployed such as protoplast fusion, CaCl₂, calcium phosphateprecipitation, electroporation, or other conventional DNA transfertechniques. The vectors may then be introduced into a second host bysimilar transfer methods, and also by cell to cell transfer methods suchit as conjugation. This cell-to-cell transfer may be accomplished usingknown techniques which depend upon the nature of the transfererbacterium, the recipient bacterium, and the cloning vector used topropagate the RT DNA. The transfer may require the use of a helperplasmid. See, for example, Ditta, G., et al., Proc. Natl. Acad. Sci.(USA) 77:7347-7351 (1980).

RT genes having DNA polymerase activity and substantially no RNase Hactivity may be obtained by deletion of deoxyribonucleotides at the 3′end of the gene which encode the portion of the polypeptide having RNaseH activity. Deletions of the RT gene may be accomplished by cutting theplasmid at selected restriction sites within the RT gene and discardingthe excised fragment. Further deletion of consecutivedeoxyribonucleotides may be accomplished by treating the fragment withan exonuclease. The DNA ends may then be joined in such a way that thetranslation reading frame of the gene is maintained. The plasmid thusobtained may then be used to transform hosts which may then be screenedfor altered RT activity. RT RNase H activity may be assayed according toGerard et al., J. Virol. 15:785-79.7 (1975). DNA polymerase activity maybe assayed according to Gerard et al,, Biochem. 13:1632-1641 (1974).Clones having DNA polymerase activity and substantially no RNase Hactivity may be used to prepare RT with altered activity.

According to these methods, the portion of the RT gene derived fromM-MLV which encodes DNA polymerase was localized to about 1495 basepairs (about 1018 to about 2512) as shown in FIG. 6. The proteinexpressed by this gene has about 503 amino acids (FIG. 6). This proteinhas DNA polymerase activity and substantially no RNase H activity.

The invention also relates to fusion proteins which comprise the reversetranscriptase of the invention. Such fusion proteins may comprise, forexample, a carrier protein which has a leader sequence of hydrophobicamino acids at the amino terminus of the reverse transcriptase. Thiscarrier protein is normally excreted through the membrane of the cellwithin which it is made. By cleavage of the hydrophobic leader sequenceduring excretion, a means is provided for producing reversetranscriptase which can be recovered either from the periplasmic spaceor the medium in which the bacterium is grown. The use of such a carrierprotein allows isolation of reverse transcriptase without contaminationby other proteins within the bacterium, and achieves production of aform of reverse transcriptase having greater stability by avoiding theenzymes within the bacterial cell which degrade foreign proteins. TheDNA and amino acid sequences for such hydrophobic leader sequences, aswell as methods of preparing such fusion proteins are taught, forexample, by Gilbert et al., U.S. Pat. No. 4,411,994 (1983).

It is also possible to prepare fusion proteins comprising the reversetranscriptase of the invention which is substituted at the amino orcarboxy termini with polypeptides which stabilize or change thesolubility of the reverse transcriptase. An amino-terminal gene fusionwhich encodes reverse transcriptase, having both DNA polymerase andRNase activity, and IME taught, for example, by Tanese, N. et al., Proc.Nat'l. Acad. Sci. 82:4944-4948 (1985). A carboxy-terminal gene fusionwhich encodes reverse transcriptase and part of the plasmid pBR322 tetgene is taught, for example, by Kotewicz, M., et al., Gene 35:249-258(19851 and Gerard, G., DNA 5:271-279 (1986).

The transformed hosts of the invention may be cultured under proteinproducing conditions according to any of the methods which are known tothose skilled in the art.

The reverse transcriptase having DNA-polymerase activity andsubstantially no RNase activity may be isolated according toconventional methods known to those skilled in the art. For example, thecells may be collected by centrifugation, washed with suitable buffers,lysed, and the reverse transcriptase isolated by column chromatography,for example, on DEAE-cellulose, phosphbcellulose (see Kotewicz et. al.,Gene 35:249-258. (198.5) or other standard isolation and identificationtechniques using, for example, polyribocytidylic acid-agarose, orhydroxylapatite or by electrophoresis or immunoprecipitation.

The reverse transcriptase so produced may be used to prepare cDNA fromRNA by, for example, hybridizing an oligo(dT) primer or othercomplementary primers with the mRNA. The synthesis of a complete cDNAmay be accomplished by adding the reverse transcriptase and all fourdeoxynucleoside triphosphates. Using the reverse transcriptase producedby the present invention allows for the preparation of cDNA from mRNAwithout concomitant degradation of the mRNA which results in incompletecDNA synthesis. The resulting RNA-DNA hybrid may be treated, forexample, with alkali or RNase H to selectively hydrolyze the RNA toleave cDNA which may be converted to double-stranded form in a secondDNA reaction catalyzed by reverse transcriptase or other DNA polymerase.See Old, R. W., et al., Principals of Gene Manipulation, second edition,Studies in Microbiology, Vol. 2, University of California Press, p. 26(1981).

The reverse transcriptase of the invention is ideally suited forincorporation into a kit for the preparation of cDNA from RNA. Such akit may comprise a carrier means being compartmentalized to receive aclose confinement therein, one or more container means, such, as vials,tubes, and the like, each of said container means comprising one of theseparate elements of the method used to prepare cDNA from RNA. Forexample, there may be provided a container means containing reversetranscriptase having DNA polymerase activity and substantially no RNaseH activity, in solution. Further container means may contain suitablebuffers, substrates for DNA synthesis such as the deoxynucleosidetriphosphate, oligo(dT) primer, and control RNA for use as a standard.

The reverse transcriptase may be present in the solution at aconcentration of 200 units/ml to 400 units/ml. The deoxynucleosidetriphosphases may be present either in lyophilized form or as part of abuffer at a concentration of 0.5 mM to 2 mM. A suitable buffer, presentat 5 times the final concentration of use, includes 250 mM Tris-HCl (pH7.5 to 8.3), 375 mM KCl, 15 mM MgCl₂, and 50 mM dithiothreitol. Theoligo (dT) may be present at a concentration of 5 μg/ml to 20 μg/ml.Control RNA, such as 2.3 kb control RNA, may be present at aconcentration of 10 μg/ml to 20 μg/ml.

The following examples are illustrative but not limiting of the methodsand compositions of the present invention. Any suitable modificationsand adaptations which are obvious to one of ordinary skill in the art inrecombinant DNA techniques are within the spirit and scope of thepresent invention.

EXAMPLES

Materials and Methods

Plasmids and Bacterial Strains

For deletion analysis of RT, a clone of M-MLV RT was constructed tooverproduce stable RT in Escherichia coli, pRT601 (FIG. 1). Gerard, G.F., et al., DNA 5:271-279 (1986). It is a pBR322-replicon containing thestrong lambda leftward promoter, pL, and the ribosome binding site ofthe lambda cII gene. (Higher copy number derivatives of pER322, such aspUC plasmids, can also be used.) The coding sequence for the RT gene wascarefully engineered into this plasmid to produce a protein with theamino terminus of the viral protein and a carboxy terminus similar tothe viral enzyme. Gerard, G. F., supra.

Two bacterial strains were used to propagate clones and express RT: K802(Maniatis, T., et al., (1982), Molecular Cloning: A Laboratory Handbook,pp. 504-505, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.),made lysogenic for lambda cIindlts857 Sam7, and N4830 (Gottesman, M. E.,et al., J. Mol. Biol. 140:57-75 (1980)), which contains a deleted acryptic lambda prophage expressing the temperature sensitive cI alleleindlts857. Bacteria were grown in super broth (SB) containing 2%tryptone, 1% yeast extract, 0.1% NaCl, pH 7.5, and 50 μg/ml ampicillin.

Plasmid Construction

Standard procedures for plasmid construction were performed as describedpreviously (Kotewicz, M. L., et al., Gene 35:249-258 (1985); Gerard, G.F., et al., DNA 5:271-279 (1986)).

Temperature Induction of E. coli Carrying pRT601 and Its Derivatives

Cultures of, bacteria were grown in SB broth overnight at 32° C. anddiluted 1:20 in fresh SB in the morning. The cells were grown at 32° C.until the A₅₉₀ was 0.8, and were induced by swirling in a 65° C. waterbath until the temperature reached 42° C. Induction was continued for 30minutes in a shaking water bath at 42° C., and then the cultures wereincubated at 37° C. and grown an additional 30 minutes.

Preparation of Cell Extracts

Unless otherwise noted, all operations were performed at 4° C. Pelletedcells from one ml of culture were washed, lysed, and centrifuged asdescribed previously (Kotewicz, M. L., supra). Supernatants were removedand assayed for RNase H and DNA polymerase activity.

Enzymes Assays

RT DNA polymerase activity in extracts was assayed specifically by usingpoly(2′-O-methylcytidylate).oligo-deoxyguanylate [(Cm)_(n).(dG)₁₂₋₁₈](Gerard, G. F., et al., DNA 5:271-279 (1986), eliminating interferencefrom cellular DNA polymerases. To establish DNA polymerase specificactivities of purified RT preparations, activity was assayed with(A)_(n).(dT)₁₂₋₁₈ (Houts, G. E., et al., J. Virol. 29:517-522 (1979) asdescribed by Gerard, G. F., et al., DNA 5:271-279 (1986). One unit ofDNA polymerase activity is the amount of enzyme that incorporates onenmole of deoxynucleoside monophosphate into acid insoluble product at37° C. in 10 min.

RNase H activity in crude extracts and purified enzyme was assayed inreaction mixtures (50 μl) containing 50 mM Tris-HCl (pH 8.3), 2 mMMnCl₂, 1 mM dithiothreitol, and [³H](A)_(n).(dT)_(n) (5 μM [³H](A)_(n),35 cpm/p-mole; 20 μM (dT)_(n)). Reactions were incubated at 37° C. for20 min and were stopped by adding 10 μl of tRNA (1 mg/ml and 20 μl ofcold 50% TCA. After 10 minutes on ice, the mixture was centrifuged for10 minutes in an Eppendorf centrifuge. Forty μl of the supernatant wascounted in aqueous scintillant. One unit of RNase H activity is theamount of enzyme required to solubilize one mole of [³H](A)_(n) in[³H](A)_(n).(dT)_(n) in 10 min at 37° C.

Synthesis of Poly(A)-Tailed RNA

Synthetic 2.3 kb and 6.2 kb RNAs containing a 19 nucleotide poly(A) tailat the 3′ end-were synthesized with T7 RNA polymerase from Xac I-cutpJD2.3- and Hind III-cut pHL3X, respectively. Reaction mixtures (0.3 ml)contained 40 mM Tris-HCl (pH 8.0), 8 mM MgCl₂, 2 mM spermidine-HCl, 5-mMdithiothreitol, 0.4 mM each of CTP, UTP, GTP, and ATP, 20 μg/ml DNA, and2,000 units/ml T7 RNA polymerase. Uniformly labeled RNA was synthesizedwith all four [α-³²P]NTPs, each at 0.4 mM and 250 cpm/pmole. After 1 hrincubation at 37° C., the RNA product was phenol extracted, ethanolprecipitated, and purified by oligo(dT)-cellulose chromatography toensure the presence of a poly(A) tail.

Conditions for cDNA Synthesis

When assessing the effect of cDNA synthesis upon the integrity oftemplate RNA, reaction mixtures (50 μl) contained 50 mM Tris-HCl (pH8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM each dATP,dGTP, and dTTP, 0.5 mM [³H]dCTP (200 cpm/pmol), 50 μg/ml (dT)₁₂₋₁₈, 20μg/ml 2.3 kb [³²P]labeled RNA, and 4,000 units/ml RT. The reactions wereincubated at 37° C. and duplicate 2.5 μl aliquots were removed at 0, 1,5, 10, 30, and 60, min. One aliquot was precipitated onto glass fiberfilters using TCA to determine the amount of cDNA synthesized, and theother aliquot was prepared for glyoxal gel analysis. Carmichael, G. G.,et al., Method. Enzymol. 65:380-391 (1980). The glyoxalated RNA wasfractionated on a 1% agarose gel, dried, and autoradiographed. In somecases, 10 units of E. coli RNase H were added to the reaction mixtureafter 60 min and the incubation continued for 10 more min beforealiquots were taken.

When measuring the ability of RT to ssynthesize a cDNA copy of long RNA,reaction mixtures (10 μl) contained the same buffer and salts, 0.5 mMeach of dATP, dGTP, dTTP, and [α-³²P]dCTP (600 cpm/pmole), 50 μg/mlactinomycin D, 50 μg/ml (dT)₁₂₋₁₈ 100 μg/ml 6.2 kb poly(A)-tailed RNA,and 20,000 units/ml RT. After 1 hr at 37° C., the product in an aliquot(1 μl) was precipitated with TCA, counted, and the remaining DNA sizefractionated on an alkaline 1.4% agarose gel according to McDonnel, M.W, et al., J. Mol. Biol. 110:119-146 (1977>.

Purification of RT

Cells were grown to an A₅₉₀ of 3 in TYN and ampicillin medium (Gerard,G. F., et al., DNA 5:271-279 (1986)) at 30° C., induced at 43° C. for 45min, and then grown at 36° C. for 3.5 hr before harvesting. RT wasextracted from 100 g of cells as described (Gerard, G. F., supra) withthe following exceptions. RT was precipitated by addition of solid(NH₄)₂SO₄ to 40% saturation. The (NH₄)₂SO₄ pellet was dissolved in 50 mlof 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M NaCl, 5% glycerol, 1 mMdithiothreitol, and 0.01% n-octyl-β-D-glucopyranoside, the suspensionwas, clarified by centrifugation at 10,000×g for 10 min, and thesupernatant was desalted on a 320 ml (5×16 cm) Sephadex G-25 column runin buffer A (20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 5%glycerol, 0.01% NP-40) plus 0.1 M NaCl. After phosphocellulosechromatography, the RT peak was pooled, diluted with an equal volume ofbuffer A, and chromatographed on a 21-ml heparin-agarose column (1.5×12cm) equilibrated in buffer A plus 0.1 M NaCl. The RT peak from theheparin-agarose column was chromatographed on a Mono-S HR 5/5 columnequilibrated in buffer A (Gerard, G. F., supra).

Results

Construction of Reverse Transcriptase Gene Deletions

Deletions of the M-MLV RT gene were constructed by cutting pRT601.(FIG. 1) at selected restriction sites within the RT gene, discardingthe excised fragment, and joining the DNA ends in such a way that thetranslation reading frame of the gene was maintained. pRTdBam-Bam wasconstructed by deleting the Bam HI fragment between nucleotide positions1,654 and 1,960 (FIGS. 1 and 2). Ligation of the Bam HI half sites atpositions 1,654 and 1,960 maintained the translation reading frameacross the site.

A deletion at the carboxy terminus of M-MLV RT (pRTdEcoRV-C) wasconstructed by deleting all of the 3′ end of the gene downstream of theEco. RV site at position 2,513 (FIGS. 1 and 2). To constructpRTd-EcoRV-C, a Sca I (position 6,238) to Eco RV (position 2,513)fragment of pRT601 containing the 5′ portion of the RT gene was ligatedto a Sca I-Eco RI fragment derived from plasmid PBRT (Gerard, G. F.,Myra). The 3,211 base pair pBRT Sca I-Eco RI fragment contained thepBR322 origin of replication and a universal translation terminatorsequence just inside the Eco RI site. The Eco RI site was repaired withDNA polymerase I Klenow fragment before ligation.

The plasmid of pRTdEcoRV-C was deposited in E. coli under the terms ofthe Budapest Treaty at the American Type Culture Collection (ATCC),Rockville, Md., and given accession number 67555.

pRT603 was constructed as described (Gerard, G. R., supra) which encodesan RT that contains 73 fewer amino acids than pRT601, all deleted fromthe carboxy terminus (FIG. 2).

DNA Polymerase and RNase H Levels in Cells Bearing Deletion Plasmids

Alteration of as little as 3 amino acids at the carboxy end of M-MLV RTcan influence markedly the stability of the protein in E. coli (Gerard,G. F. supra). This must be taken into consideration in makingcorrelations between cloned RT deletions and enzymatic activities in E.coli extracts. Both DNA polymerase and RNase. H activity must be assayedand relative enzyme levels compared. For example, pRT603 codes for an RTwith 73 fewer amino acids at the carboxy terminus than pRT601 RT(Gerard, G. F., supra; FIG. 2). The level of DNA polymerase activity inE. coli extracts of pRT603 RT is reduced 5 fold relative to pRT601.(Gerard, G. F. supra). However, the DNA polymerase and RNase H specificactivities of purified pRT601 and pRT603 RT are comparable (Table 2).The reduced DNA polymerase activity in E. coli extracts of pRT603 RT isnot due to a selective effect of the deletion on DNA polymeraseactivity, but rather to a reduction in the stability of pRT603 RTrelative to pRT601 RT in cells (t_(1/2) of 7 min versus 33 min) (Gerard,G. F. supra). Therefore, deletions within 70 amino acids of the RTcarboxy terminus do not affect either RNase H or DNA polymeraseactivity.

In contrast, the DNA polymerase activity of pRTdBam-Bam RT waseliminated totally without affecting RNase H activity (Table 1) by thedeletion of 102 amino acid residues between amino acids 212 and 314(FIG. 2). Introduction of a more extensive deletion of 180 amino acidsat the carboxy end of RT in pRTdEcoRV-C RT (FIG. 2) yielded extractswith RT DNA polymerase levels unchanged compared to pRT601 extracts, butwith RNase H levels reduced 7.5 fold (Table 1). The residual RHase Hactivity in pRTdEcoRV-C extracts could be due to E. coli RNase H, the5′→3′ exonuclease of DNA polymerase I, or a small amount of residualRT-coded RNase H activity. To resolve this issue, pRTdEcoRV-C RT waspurified and compared to RT encoded by pRT601.

Purification and Properties of pRTdEcoRV-C RT

M-MLV reverse transcriptase encoded by pRTdEcoRV-C, pRT601, and pRT603were purified as described in Materials and Methods. A summary of thepurification of pRTdEcoRV-C RT is presented in Table 3. Three columnsteps produced a nearly homogeneous mutant enzyme with the same DNApolymerase specific activity as pRT601 RT with the template-primer(Cm)_(n).(dG)₁₂₋₁₈ (Table 2). With (A)_(n).(dT)₁₂₋₁₈, the mutant enzymehad one-fourth the DNA polymerase activity of pRT601 RT (Table 2). RNaseH activity of purified pRTdEcoRV-C RT was undetectable using[³H](A)_(n).(dT)_(n) as the substrate. Most RNase H activity in extractswas eliminated from mutant RT by precipitation of the enzyme with 40%(NH₄)₂SO₄ (Table 3). Under these conditions, DNA polymerase I remainssoluble (Richardson, C., et al., J. Biol. Chem. 239:222-230 (1964)), asdoes most of the RNase H activity in the extract. As judged bySDS-polyacrylamide gel electrophoresis, pRTdEcoRV-C RT purified throughthe Mono-S column was greater than 90% pure and had a molecular weightof 56,000 (FIG. 3), consistent with the molecular weight (57,000)predicted by the DNA sequence.

A number of enzymatic properties of purified pRTdEcoRV-C RT and pRT601RT were compared and were found to be similar. These included half lifeat 37° C., monovalent and divalent metal ion optima, fidelity of dNTPincorporation with homopolymer templates, and insensitivity tostimulation by polyanions. The abilities of the two enzymes tosynthesize heteropolymeric DNA were also compared. FIG. 4 shows thatpRTdEcoRV-C RT catalyzed the synthesis of full-length cDNA from 6.2 kbRNA more efficiently than pRT601 RT. The amount of cDNA synthesized from1 μg of RNA was 0.28 μg (34% full-length) and 0.24 μg (24% full-length)with pRTdEcoRV-C RT and pRT601 RT, respectively.

To confirm that pRTdEcoRV-C RT completely lacked, RNase H activity, theintegrity of a uniformly ³²P-labeled RNA template after conversion tohybrid form during RT-catalyzed DNA synthesis was examined. FIG. 5 showsthat with pRT601 RT, the full-length 2.3 kb RNA template was degradedtotally after 5 min of synthesis In contrast, with pRTdEcoRV-C RT theRNA was intact even after 60 min. The amount of cDNA synthesized after60 min from 1 μg of RNA was 0.67 and 0.76 μg with pRT601 and pRTdEcoRV-CRT, respectively. When 10 units of E. coli RNase H were added to thepRTdEcoRV-C RT reaction after 60 min of incubation, all of the RNA wasdegraded, confirming the hybrid state of the RNA. In addition, 15 μg(1,200 units) of pRTdEcoRV-C RT solubilized no radioactivity from a[³H](A)_(n).(dT)_(n) substrate in which the [³H](A)_(n) had a specificactivity of 2,200 cpm/pmole (Materials and Methods).

Experiments with a frameshift mutant of MLV producing a 47K RT moleculetruncated at the carboxy terminus (Levin, J. G. et al., J. Virol.51:470-478 (1984)) and with antibodies to synthetic peptides modeled toRous sarcoma virus pol gene sequences (Grandgenett, D. et al., J. Biol.Chem. 260:8243-8249 (1985)) suggest the RNase H activity of RT resideswithin the amino-terminal portion of the molecule. Conversely, theextensive homology found between the amino acids of E. coli RNase H andthe 153-residue segment at the carboxy-terminal end of M-MLV RT(Johnson, M. S. et al. Proc Natl. Acad. Sci. (USA) 83:7648-7652 (1986))suggests the RNase H activity resides within the carboxy-terminalportion of RT.

By deleting large segments (100 to 200 codons) of the M-MLV RT gene, theregions within the RT molecule responsible for DNA polymerase and RNaseH activity have been identified. DNA polymerase was mapped to the aminohalf of the molecule, and RNase H to within 200 amino acids of thecarboxy end, confirming the predictions based upon amino acid homology(Johnson, M. S. et al., supra). In this context, the results with one RTclone, pRT603. (FIG. 2), are of interest. The RT protein encoded bypRT603 is missing the carboxy half of the 153 amino acid segment of RThomologous to E. coli RNase H, which includes 20 of 48 homologous aminoacids. Yet, pRT603 RT has normal levels of RNase H activity. Thesemissing, homologous residues apparently are not required for catalysis,and might serve a nucleic acid binding or structural role. Consistentwith the latter, a single amino acid change at a position 12 residuesfrom the carboxy end of E. coli RNase H produces a 10-fold reduction inRNase H specific activity (Kanaya, S. et al., J. Bacteriol.154:1021-1026 (1983)). This reduction appears to be the result ofaltered protein conformation (Kanaya, S. et al., supra).

If the RT polymerase and nuclease active sites reside on separatestructural domains, it should be possible theoretically to isolate twoseparate protein fragments, each with a single activity. A 24K to 30Kproteolytic fragment of RT possessing only RNase H activity has beenisolated (Lai, M. H. T. et al., J. Virol. 25:652-663 (1978); Gerard, G.F., J. Virol. 26:16-28 (1978); Gerard, G. F., J. Virol. 37:748-754(1981)), but unfortunately, the location of the RNase H fragment in theparent RT polypeptide has not been established, and no analogous DNApolymerase containing fragment has ever been found. The resultspresented here show that of the 684 amino acids in pRT601 RT, residuesbetween amino acid 212 and 314 are required for DNA polymerase activity,and residues between amino acid 503 and 611 are required for RNase Hactivity. They also demonstrate for the first time that the RT DNApolymerase activity can exist independently of RNase H activity on an RTprotein fragment. Purified pRTdEcoRV-C RT appeared to be totally devoidof RNase H activity, based upon two sensitive assays, and to have fullDNA polymerase activity. However, these results do not rule out thepossibility that the two active centers share a portion(s) of the RTmolecule.

Demonstration of a separate structural domain for the RNase H activecenter was attempted by constructing two amino-terminal deletionderivatives of pRT601. The first derivative contained sequences for theEco RV site at position 2513 to the ₃′ end of the RT gene (see FIG. 2),and the second contained sequences from an Mco I site at position 2302to the 3′ end of the RT gene. Unfortunately, neither clone produceddetectable RNase H activity in E. coli crude extracts. Such negativeresults are difficult to interpret because the proteins might be unableto fold in an active form, or might be extremely labile.

Deletion of the carboxyterminal one-fourth of the M-MLV RT molecule didnot disrupt the ability of the protein to fold in an activeconformation. pRTdEcoRV-C. RT copied heteropolymeric RNA, moreefficiently than intact RT. Yields of cDNA from 1 μg of 2.3 kb and 6.2kb RNA were 0.76 μg (50% full-length) and 0.28 μg (34% full-lengthy,respectively. Also, the truncated and intact enzymes had the same DNApolymerase specific activity with (Cm)_(n).(dG)₁₂₋₁₈. However, thetruncated enzyme copied (A)_(n).(dT)₁₂₋₁₈, only one fourth asefficiently as the parent RT. The origin of this difference has not yetbeen established. TABLE 1 DNA polymerase and RNase H activity inextracts of heat induced E. coli F802 (lambda) bearing pRT601 or one ofits derivatives. DNA polymerase RNase H Activity^(a) Activity^(b) (cpmincorporated/ (cpm solubilized/ Plasmid 2.5 μl extract) 2.5 μl extract)pRT601 10,977 2,020 pRTdBam-Bam 179 1,564 pRTdEcoRV-C 10,038 268^(a)Reverse transcriptase DNA polymerase activity was assayed with(Cm)_(n) · (dG)₁₂₋₁₈ (Materials and Methods).^(b)RNase H activity was assayed with [³H](A)_(n) · (dT)_(n) (Materialsand Methods).

TABLE 2 Comparison of activities of purified RT coded by pRT601, pRT603,and pRTdEcoRV-C DNA polymerase Activity with (Cm)_(n) · (dG)₁₂₋₁₈(A)_(n) · (dT)₁₂₋₁₈ RNase H Activity Enzyme (Units/mg) (Units/mg)(Units/mg) pRT601 21,700 350,000 2,670 pRT603 ND^(a) 230,000 1,100pRTdEcoRV-C 17,500 81,000 —^(b)^(a)ND, not determined^(b)No activity was detected

TABLE 3 Summary of the purification of pRTdEcoRV-C RT DNA PolymeraseActivity^(a) RNase H Activity Total Specific Specific Protein^(c) TotalActivity Yield Total Activity Yield Fraction (mg) (Units) × 10³(Units/mg) × 10³ (%) (Units) × 10³ (Units/mg) × 10³ (%) Crude lysate7,913 255 0.03 100 80 0.01 100 Polymin P Supernatant 2,735 323 0.12 127157 0.06 196 (NH₄)₂SO₄ pellet 63 168 1.38 66 6.0 0.10 7 Phosphocellulosepool 8.8 167 19.0 66 2.0 0.23 3 Heparin-agarose pool 6.5 111 17.1 44—^(b) — — Mono S pool 3.1 55 17.5 22 —^(b) — —^(a)DNA polymerase activity was assayed with (Cm)_(n) · (dG)₁₂₋₁₈^(b)No activity could be detected^(c)Protein concentrations were determined using bovine serum albumin asstandard according to Lowry, O. H., et al., J. Biol. Chem. 239: 222-230(1964).

1. A gene which encodes reverse transcriptase having DNA polymeraseactivity and substantially no RNase H activity. 2-23. (canceled)